Many metals pose a risk as environmental contaminants. A well-known example is lead. Low level lead exposure can lead to a number of adverse health effects, with as many as 9-25% of pre-school children presently at risk. The level of lead in the blood considered toxic is xe2x89xa710 xcexcg/dL (480 nM). Current methods for lead analysis, such as atomic absorption spectrometry, inductively coupled plasma mass spectrometry, and anodic stripping voltammetry, often require sophisticated equipment, sample pre-treatment, and skilled operators.
Simple, rapid, inexpensive, selective and sensitive methods that permit real time detection of Pb2+ and other metal ions are very important in the fields of environmental monitoring, clinical toxicology, wastewater treatment, and industrial process monitoring. Furthermore, methods are needed for monitoring free or bioavailable, instead of total, metal ions in industrial and biological systems.
Fluorescence spectroscopy is a technique well suited for very small concentrations of analytes. Fluorescence provides significant signal amplification, since a single fluorophore can absorb and emit many photons, leading to strong signals even at very low concentrations. In addition, the fluorescence time-scale is fast enough to allow real-time monitoring of concentration fluctuations. The fluorescent properties only respond to changes related to the fluorophore, and therefore can be highly selective. Furthermore, fluorometers for uses in the field are commercially available. Fluorescent detection is also compatible with fiber-optic technology and well suited for in vivo imaging applications. Several fluorescence-related parameters can be assessed for the purpose of sensing, including fluorescence intensity, emission or excitation wavelength, fluorescence lifetime and anisotropy.
Many fluorescent chemosensors, including fluorophore-labeled organic chelators (Rurack, et al., 2000; Hennrich et al., 1999; Winkler et al., 1998; Oehme and Wolfbeis, 1997) and peptides (Walkup and Imperiali, 1996; Deo and Godwin, 2000; Pearce et al., 1998), have been developed for metal ion detection. These ion sensors are usually composed of an ion-binding motif and a fluorophore. Metal detection using these fluorescent chemosensors relies on the modulation of the fluorescent properties of the fluorophore by the metal-binding event. Detection limits on the level of micromolar and even nanomolar concentrations have been achieved for heavy metal ions including Zn2+, Cu2+, Hg2+, Cd2+ and Ag+. The design and synthesis of a chemosensor that exhibits highly selective and sensitive binding of the metal ion of choice in aqueous solution is still a big challenge, although the metal binding and the fluorescent moieties of the sensor can be systematically varied to achieve desired properties.
Nucleic acid molecules have previously been adapted to sense the presence of nucleic acids and to detect gene mutations from inherited diseases or chemical damages. In recent years, the molecular recognition and catalytic function of nucleic acids have been extensively explored. This exploration has lead to the development of aptamers and nucleic acid enzymes.
Aptamers are single-stranded oligonucleotides derived from an in vitro evolution protocol called systematic evolution of ligands by exponential enrichment (SELEX). Nucleic acid aptamers have been isolated from random sequence pools and can selectively bind to non-nucleic acid targets, such as small organic molecules or proteins, with affinities as high as 10xe2x88x9214 M (Uphoff et al., 1996; Famulok, 1999). Most aptamers undergo a conformational change when binding their cognate ligands. With this property, several DNA and RNA aptamers have been engineered to sense L-adenosine or thrombin through an internally labeled fluorescent reporter group (Jhaveri et al., 2000). Thus, the conformational change in the aptamer upon binding leads to a change in fluorescence.
Nucleic acid enzymes are nucleic acid molecules that catalyze a chemical reaction. In vitro selection of nucleic acid enzymes from a library of 1014-1015 random nucleic acid sequences offers considerable opportunity for developing enzymes with desired characteristics (Breaker and Joyce, 1994; Breaker, 1997). Compared with combinatorial searches of chemo- and peptidyl-sensors, in vitro selection of DNA/RNA is capable of sampling a larger pool of sequences, amplifying the desired sequences by polymerase chain reactions (PCR), and introducing mutations to improve performance by mutagenic PCR.
Allosteric ribozymes (or aptazymes), which combine the features of both aptamer and catalytic RNA, also hold promises for sensing small molecules (Potyrailo el al., 1998; Koizumi et al., 1999; Robertson and Ellington, 1999, 2000). Their reactivity is modulated through the conformational changes caused by the binding of small organic molecules to an allosteric aptamer domain. Therefore, the signal of ligand binding can be transformed into a signal related to chemical reaction.
Divalent metal ions can be considered as a special class of cofactors controlling the activity of nucleic acid enzymes. The reaction rate of the nucleic acid enzymes depends on the type and concentration of the metal ion in solution. Several RNA and DNA enzymes obtained through in vitro selection are highly specific for Cu2+, Zn2+, and Pb2+, with metal ion requirements on the level of micromolar concentrations (Breaker and Joyce, 1994; Pan and Uhlenbeck, 1992; Carmi et al., 1996; Pan et al., 1994; Cuenoud and Szotak, 1995; Li et al., 2000; Santoro et al., 2000).
The present invention uses nucleic acid enzymes as signal transducers for ion detection. Compared with fluorescent chemosensor and protein biosensors, nucleic acid-based sensors are more amenable to combinatorial search for sequences with desired metal specificity and affinity. In addition, DNA, in particular, is stable and can be readily synthesized. A wide range of fluorescent dyes can be easily introduced at specific sites to suit different needs. DNA-based biosensors can also be adapted for use with optical fiber and DNA-chip technology for applications such as in vivo imaging, in situ detection, and array sensing.
In one aspect, the present invention provides for specific and sensitive biosensors of ions. The biosensors are useful in methods of detecting the presence of an ion, particularly metal ions such as Pb2+. In certain embodiments, the biosensors may be used to determine the concentration of a particular ion in a solution.
The biosensors of the present invention use nucleic acid enzymes that require the presence of specific ions for their activity. Enzymatic activity leads to hydrolytic cleavage of a substrate nucleic acid that may be part of the nucleic acid enzyme itself The resulting cleavage product then may be detected indicating the presence of the ion.
In a preferred embodiment, the biosensor comprises a fluorophore and a quencher arranged in proximity such that prior to cleavage the fluorescence intensity is decreased by the quencher. However, upon cleavage, the fluorophore and quencher are separated leading to an increase in fluorescence intensity. In a further preferred embodiment, the biosensor contains an array of nucleic acid enzymes having a range of sensitivities and specificities to several different ions.
A xe2x80x9cnucleic acid enzymexe2x80x9d is a nucleic acid molecule that catalyzes a chemical reaction. The nucleic acid enzyme may be covalently linked with one or more other molecules yet remain a nucleic acid enzyme. Examples of other molecules include dyes, quenchers, proteins, and solid supports. The nucleic acid enzyme may be entirely made up of ribonucleotides, deoxyribonucleotides, or acombination of ribo- and deoxyribonucleotides.
A xe2x80x9csamplexe2x80x9d may be any solution that may contain an ion (before or after pre-treatment). The sample may contain an unknown concentration of an ion. For example, the sample may be paint that is tested for lead content. The sample may be diluted yet still remain a sample. The sample may be obtained from the natural environment, such as a lake, pond, or ocean, an industrial environment, such as a pool or waste stream, a research lab, common household, or a biological environment, such as blood. Of course, sample is not limited to the taking of an aliquot of solution but also includes the solution itself. For example, a biosensor may be placed into a body of water to measure for contaminants. In such instance, the sample may comprise the body of water or a particular area of the body of water. Alternatively, a solution may be flowed over the biosensor without an aliquot being taken. Furthermore, the sample may contain a solid or be produced by dissolving a solid to produce a solution. For example, the solution may contain soil from weapon sites or chemical plants. xe2x80x9cMeasuring the product of the nucleic acid enzymatic reactionxe2x80x9d includes measuring the result of the production of a product by an enzyme. For example, in an embodiment where the substrate comprises a quencher and the enzyme comprises a fluorophore and cleavage of the substrate by the enzyme leads to dissociation of the product from the enzyme, xe2x80x9cmeasuring the productxe2x80x9d includes detecting the increase of fluorescence. Thus, one is measuring the product by detecting its inability to quench fluorescence.
xe2x80x9cContacting a nucleic acid enzyme with a samplexe2x80x9d includes placing the sample and enzyme in proximity such that an ion in the sample could be used as a cofactor. xe2x80x9cContactingxe2x80x9d includes such acts as pipetting a sample onto a solid support or into a tube or well containing the nucleic acid enzyme. Alternatively, the enzyme may be brought to the sample. For example, the enzyme may be placed into a stream to monitor for the presence of a contarninant.